This proposal is to determine the role of cdk5 protein kinase in neuronal differentiation and the role of Rho family of GTPases in this process. Research is based on the applicant's recent observations concerning a cyclin-dependent protein kinase, cdk5, she helped isolate and characterize as a postdoctoral fellow. Two observations she has made since she became an independent investigator, and preliminary data, have led to applicant to hypothesize that cdk5 is involved in neuron migration and neurite outgrowth, especially with respect to the development of laminar structures such as the neocortex. The present proposal is based on two published observations in the applicants laboratory and on recent preliminary data showing that the cdk5 kinase interacts with the Rho subfamily of GTPases. The first observation is that knock outs of the 35-kDa regulator of cdk5-protein kinase, p35, produces a markedly abnormal cortex; many neurons migrate to an inside- out position, reminiscent of the Reeler mouse. The second is that cultured cortical embryonic neurons develop abnormally short neurites when transfected with dominate negative forms of cdk5 kinase or its regulator. p35. Extensive preliminary results are presented using transfection of cultured cells that support a role for p35/cdk5 in morphology regulation and indicating that the Rho subfamily of GTPases is involved in this regulation. The applicant shows that transfection expression of p35 in fibroblasts induces morphological changes and that co-transfection of p35 and the Rho GTPases co-localize in fibroblasts when examined by immunocytochemistry. When co-transfected, tagged Rho GTPases and p35/cdk5 co-purify from lysates of the expression cells, showing a direct physical interaction of these proteins. This is the basis for the hypothesis that the Rho GTPases mediate the p35/cdk5 protein kinase influence on cell morphology. In addition, a yeast two hybrid screen isolated several additional proteins that interact with the p35/cdk5 kinase. Three of these are proposed to be further analyzed. Two extensive specific aims are proposed. The first is to investigate the nature of the interaction between p35/cdk5 and the Rho family of GTPases and the consequence of their interaction. The second is to characterize the three novel proteins which interact with p35/cdk5 and explore the functional consequences of these interactions. The overall goal is to understand the molecular mechanisms by which neuronal migration is regulated. To investigate the nature of the interactions between p35/cdk5 and Rho GTPases, a series of transfection assays will be carried out. Normal or mutated cdk5 and normal or mutated GTPases will be transfected into Cos cells. Their interaction with one another will be assessed in the intact cells using immunocytochemistry. This will be further analyzed in lysates by immunoprecipitation and/or purification of tagged proteins from the lysed cells followed by western blotting using a variety of immunological probes. To assess biochemical consequence of the interaction of cdk5 with GTPases the influence of these structural mutants will also be analyzed for their effects on several putative protein targets of the p35/cdk5. To assess biological consequences of the interaction between p35/cdk5 and GTPases, the structural mutants found to be interesting in the transfection studies will analyzed for their influence on neurites formation, in cortical cell cultures, and on the extension of neurites in a three dimensional manner in in vitro brain slices. The mutants will be administered to the brain slices using a rabies viral vector with the help of Dr. Rachel Neve. The second specific aim proposes to carry out studies similar to those described above, but using transfection of each of the three proteins the applicant has found to interact with cdk5 in the two hybrid screen assay.